The present invention relates generally to the field of chemotherapy and more particularly to the invention of novel anti-neoplastic agents denominated xe2x80x9chydroxyphenstatinxe2x80x9d and selected prodrugs thereof.
The elucidation and isolation of agents from the African bush willow combretum caffrum first identified combretastatin A-4 as described in U.S. Pat. No. 4,996,237 which issued to G. R. Pettit et al., on Feb. 26, 1991. Other early efforts to develop a combretastatin A-4 prodrug are described in U.S. Pat. No. 5,561,122, which issued to G. R. Pettit on Oct. 1, 1996. The general background information from each of these patents is incorporated herein by this reference thereto.
The potent cancer cell growth and tubulin assembly inhibitor combretastatin A-4 was originally isolated from the African tree Combretum caffrum (Combretaceae) circa 1985 and has been undergoing preclinical development since that time. However, because of the very limited aqueous solubility of the phenol and its alkali metal salts, drug formulation attempts gave unsatisfactory results. Accordingly, the present disclosure comprises a benchmark in the continuing effort to synthesize practical water soluble prodrugs based on combretastatin A-4 and is a significant and remarkably unexpected extension of those early efforts which are described in U.S. Pat. No. 5,561,122, supra.
The African willow tree Combretum caffrum Kuntze (Combretaceae) has proven to be a very productive source of cancer cell growth (murine P388 lymphocytic leukemia) inhibitory stilbenes, bibenzyls and phenanthrenes. Since 1979 promising leads have been pursued which were focused on the three most active (inhibition of cancer cell growth and polymerization of tubulin (Id.)) constituents, namely combretastatin A-1 (3a), A-2 (4), and A-4 (3b) (Id.) (See, FIG. 1). Of these, combretastatin A-4 (3b) has reached the most advanced stage of preclinical development as the very soluble prodrug 3c. Meanwhile other research groups have also been further extending structure/activity relationships (hereinafter referred to as xe2x80x9cSARxe2x80x9d) among the combretastatins and related stilbenes.
The formation of new blood vessels, known as xe2x80x9cangiogenesisxe2x80x9d (neovascularisation), is controlled by a very complex series of biochemical interactions involving a large number of angiogenic factors ranging from various cytokines (e.g. IL-1) and growth factors (e.g., GM-CSF) to serine proteases (e.g. urokinase). In general, normal angiogenesis involves the activation and transport of endothelial cells from already formed blood vessels to new locations. Normally that transition takes three (3) months to a year except in wound healing and in certain stages of female reproductive biology. When the angiogenic control mechanisms fail, the results lead to a wide range of human disease categories, such as cancer, psoriasis, hemangioma, atherosclerotic plaque, diabetic and macular retinopathy, neovascular glaucoma, and vascular adhesions following surgery.
Because the African bush willow (Combretum caffrum) constituents, (Isolation, Structure and Synthesis of Combretastatin A-1 and Combretastatin B-1, Potent New Inhibitors of Microtubule Assembly, Derived from Combretum caffrum. J. Nat. Prod. 1987, 50, 119-131), combretastatins A-1 (3a) and A-4 (3b) were isolated and designated as well as their phosphate prodrug; (Pettit, G. R.; Rhodes, M. R. Antineoplastic Agents 389. New Syntheses of Combretastatin A-4 Prodrug. Anti-Cancer Drug Des. 1998, 13, 183-191; and Pettit, G. R.; Lippert, J. W. III. Antineoplastic Agents 429. Synthesis of Combretastatin A-1 and Combretastatin B-1Prodrugs. Anti-Cancer Drug Des. 1999, in preparation.), derivatives (3c,e) which displayed very promising antineoplastic, cancer antiangiogenesis. Recently extended SAR investigations of these cis-stilbenes have been conducted. Indeed, combretastatin A-4 prodrug (3c) has been undergoing a series of phase I human cancer clinical trials since November 1998.
Previous SAR analyses of the combretastatin A-4 series have indicated that the cis configuration of the stilbene unit is the most important factor for inhibition of cancer cell growth and inhibiting tubulin assembly. With the corresponding (E)-stilbenes, the cancer cell growth inhibitory and antitubulin activity is greatly reduced from that exhibited by the corresponding (Z)-isomers. Initially, both the trans-isomers and were found to be moderately active as cancer cell growth inhibitors. Later studies using trans-stilbene revealed that freshly prepared solutions in dimethyl sulfoxide were inactive and only gained activity with the passage of time suggesting that the trans-isomers were slowly converted to the cis active isomer.
Furthermore, a structure-activity relationship (SAR) study of the South African willow tree (Combretum caffrum) antineoplastic constituent combretastatin A-4 (3b) led to the discovery of a potent cancer cell growth inhibitor designated phenstatin (5a). This benzophenone derivative of combretastatin A-4 showed great antineoplastic activity and the benzophenone derivative of combretastatin A-1 was synthesized. The benzophenone, designated hydroxyphenstatin (6a), was synthesized by coupling of a protected bromobenzene and a benzaldehyde to give the benzhydrol with subsequent oxidation to the ketone. Hydroxyphenstatin was converted to the sodium phosphate prodrug (6e) by a dibenzyl phosphite phosphorylation and subsequent benzyl cleavage (6axe2x86x926dxe2x86x926e). Hydroxyphenstatin (6a) was a potent inhibitor of tubulin polymerization comparable to combretastatin A-1 (3a).
Podophyllum, the roots and rhizomes of Podophyllum species such as Peltatumi L. (Podophyllaceae, May Apple) found important uses including cancer and antiviral applications in the traditional medicine of early Americans and in India. Indeed, it was an important component of the U.S. Pharmacopoeia from 1820-1942 (the derived resin has been found to contain up to 38% podophyllotoxin (1a) and was the first terrestrial plant anticancer agent developed to clinical trials by the U.S. National Cancer Institute some fifty years ago. Subsequently, podophyllotoxin has been converted to the glycoside derivative known as etoposide (1b), now widely used in human cancer treatment.
In 1958, a SAR investigation was initiated which focused on the trimethoxy and methylenedioxy diarylmethylene unit of podophyllotoxin (1a). While not detected at the time, owing to limitations of the early antineoplastic evaluation options, it was later found that the diarylketone (2) significantly inhibited the growth of the P388 lymphocytic leukemia cell line with an ED50 value of 2.6 xcexcg/ml. By 1978, while investigating the cancer cell growth inhibition of the African willow tree Combretum caffrum Kuntze (Combretaceae) three potentially important constituents were discovered which were designated combretastatins A-1 (3a), A-2 (4), and A-4 (3b). Combretastatin A-4, as the water soluble prodrug (3c), subsequently reached the most advanced stage of preclinical and clinical development. More recently, the diarylketone named phenstatin (5a) was discovered (See: Pettit, G. R.; Toki, B.; Herald, D. L.; Verdier-Pinard, P.; Boyd, M. R.; Hamel, E.; Pettit, R. K. Antineoplastic Agents 379. Synthesis of Phenstatin Phosphate. J. Med Chem. 1998, 41, 1688-1695) which was found to be structurally related to podophyllotoxin (1a) and combretastatin A-4 (3b) and proved to be a very strong anticancer substance comparable to stilbene (3b). These and other results (See: Pettit, G. R.; Lippert, J. W. III; Boyd, M. R.; Hamel, E.; Pettit, R. K. Antineoplastic Agents 442. The Remarkable Antitubulin Assembly and Cancer Cell Growth Inhibition of (4S,5S)-4-(2xe2x80x3,3xe2x80x3-dihydroxy-4xe2x80x3-methoxyphenyl)-5-(3xe2x80x2,4xe2x80x2,5xe2x80x2-trimethoxyphenyl)-1,3-dioxolane. J. Med Chem. in preparation) encouraged efforts to undertake the synthesis and evaluation of diphenol (6a).
The general procedure reported in 1962 for obtaining ketone (2) (See: Pettit, G. R.; Baumann, M. F.; Rangammal, K. N. Antineoplastic Agents V. The Aromatic System of Podophyllotoxin (Part B). J. Med Pharm. 1962, 5, 800-808) was attempted first. Coupling reactions between 2,3-bis(t-butyldimethylsilyloxy)-4-methoxy-bromobenzene (7b) and N-(3,4,5-trimethoxybenzoyl)morpholine (8a) utilizing either n-butyl- or tert-butyllithium were unsuccessful. Changing the acylating agent to a benzoyl chloride was also not productive. Presumably, the bulky TBDMS substituents caused enough steric hindrance to prevent nucleophilic attack of the lithium-benzene complex on the carbonyl group. Thus, the smaller methoxymethyl ether (MOM) protecting group was next chosen. (See: Greene, T. W.; Wutz, P. G. M. Protective Groups in Organic Synthesis. J. Wiley and Sons: New York, 1999; pp. 27-33). However, formation of the benzophenone using the MOM-protected bromobenzene (7c) and either the morpholine amide (8a) or the benzoyl chloride (8b) met only with limited success, affording 24% and 20% yields, respectively. Further attempts to prepare protected diphenol (6c) using Grignard reactions, Weinreb amides, (See: Nahm, S.; Weinreb, S. M. N-Methoxy-N-methylamides as Effective Acylating Agents. Tetrahedron Lett. 1981, 22, 3815-3818), and dimethylamides also afforded low yields (14-44%). Application of organometallic reagents such as La(OTf)3, Bu3P and Fe(acac)3 did not provide improved yields of ketone (6c).
In order to determine if the protecting groups were interfering, ketone formation was evaluated starting with 2,3,4-trimethoxybromobenzene (9) and morpholine amide (8a). The resulting yields were found to range from 17-20%. These results indicated that the protecting groups used in the preceding reaction may not have significantly influenced the poor yields. Later, however, it was found that condensing the bromobenzene (9) with 3,4,5-trimethoxybenzaldehyde led to the formation of benzhydrol (14) in 86% yield. Subsequent oxidation with pyridinium dichromate (PDC) to benzophenone (13) provided 83% yield. These favorable results led to the utilization of the efficient reaction between an aldehyde and an organolithium reagent to prepare a benzhydrol derivative of ketone (6a). This approach was realized when the lithium derivative of MOM-protected bromobenzene (7c) and 3,4,5-trimethoxybenzaldehyde were condensed to afford protected benzhydrol (15) in 92% yield. Oxidation of protected benzhydrol by PDC produced protected hydroxyphenstatin (6c) in good yield (96%) and MOM-cleavage (acidic) afforded hydroxyphenstatin (6a) in 97% yield. It is toward these discoveries and the unexpected results obtained therefrom that the present disclosure is directed.
A structure-activity relationship (SAR) study of the South African willow tree (Combretum caffrum) antineoplastic constituent combretastatin A-4 (3b) led to the discovery of a potent cancer cell growth inhibitor designated phenstatin (5a). This benzophenone derivative of combretastatin A-4 showed great antineoplastic activity and the benzophenone derivative of combretastatin A-1 was likewise synthesized. This, benzophenone, designated xe2x80x9chydroxyphenstatinxe2x80x9d (6a), was synthesized by coupling of a protected bromobenzene and a benzaldehyde to give the benzhydrol with subsequent oxidation to the ketone. Hydroxyphenstatin was converted to the sodium phosphate prodrug (6e) by a dibenzyl phosphite phosphorylation and subsequent benzyl cleavage (6axe2x86x926dxe2x86x926e). Hydroxyphenstatin (6a) was found to be a potent inhibitor of tubulin polymerization comparable to combretastatin A-1 (3a).
A principal object of the present invention is the synthesis, elucidation of structure and utilization of a novel anti-neoplastic compound obtained while attempting to synthesize anticancer substances related to combretastatin A-1, obtained from the South African willow tree (Combretum caffrum) and found to obtain greater potency, enhanced solubility and less adverse side effects than had been previously obtained from other compounds previously derived therefrom.
Another object of the present invention is the isolation, elucidation and utilization of a synthetic derivative of the potent cell growth inhibitor designated xe2x80x9cphenstatinxe2x80x9d in a continuing effort to develop synthetic agents capable of inhibiting the spread of cancer cells in the human environment with minimal side effects.
These and still further objects as shall hereinafter appear are readily fulfilled by the present invention in a remarkably unexpected manner as will be readily discerned from the following detailed description of an exemplary embodiment thereof.